The goal of this proposal is to determine the structural basis by which G protein-coupled receptors (GPCRs) activate specific G proteins. The majority of hormones and neurotransmitters communicate information to cells via GPCRs, and GPCRs represent the largest group of targets for drug development. Our laboratories have a long-standing interest in elucidating the structure and mechanism of G protein activation by GPCRs. During the previous funding period we succeeded in obtaining the first crystal structure of a GPCR-G protein complex: the beta2 adrenoceptor (?2AR) in complex with Gs, the stimulatory protein for adenylyl cyclase. This structure provides important mechanistic insight into G protein activation, but at the same time raises new questions that will be addressed in this competitive renewal. Specific Aims include: Aim 1. Determine the structural basis of GPCR-G protein coupling specificity. The structure of the ?2AR-Gs complex provided the first high-resolution insights into transmembrane signaling by a GPCR. However, additional GPCR-G protein complexes will be required to understand the structural basis for G protein coupling specificity, and to determine if the mechanistic insights obtained from the ?2AR -Gs structure are generalizable to other GPCR-G protein pairs. We therefore propose to obtain three additional GPCR-G protein complex structures: (1) the vasopressin receptor-Gs complex; (2) the structure of the ?2AR-Gi complex; and (3) the structure of the M2R-Gi complex. Aim 2. Characterize the formation of the ?2AR-Gs complex from the GDP bound Gs heterotrimer. The ?2AR- Gs crystal structure represents a single state in a complex cycle of events. The process of complex formation and dissociation remains poorly understood. These are dynamic process that may not be addressable by crystallography; however, the ?2AR-Gs structure will provide the basis for designing and interpreting biochemical and biophysical studies to characterize the mechanism of complex formation and dissociation. In Aim 2 we will characterize the low affinity interactions between the ?2AR and GDP bound Gs. These interactions may play a role in G protein coupling specificity. Aim 3. Characterize the process of ?2AR -Gs dissociation following GTP binding. The goal of this Aim is to understand how the ?2AR -Gs complex dissociates into active signaling proteins upon binding GTP and to identify persisting interactions between any of the three components: ?2AR, G?s and G??. Aim 4. Characterize the dynamic behavior of the G?s alpha helical domain. The most surprising and unexpected feature of ?2AR-Gs structures is the flexible link between the two domains that make up G?s: the Ras-like GTPase domain and the alpha helical domain (AHD). This subaim will further characterize the interactions between these two domains in the ?2AR-Gs complex as well as in GTP and GDP bound states. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to determine the mechanism by which G protein coupled receptors (GPCRs) activate specific cellular G proteins in response to hormones and neurotransmitters, and modify the function of cells. This information will facilitate the process of drug discovery for GPCRs, which are the largest family of membrane proteins in the human genome. Drugs acting on GPCRs can have an impact on a broad spectrum of diseases including: cardiovascular disease, pulmonary disease, inflammation, diabetes and obesity, behavioral disorders and Alzheimer's disease.